Shark Antibody Research Articles

Preparation of pH-sensitive nanogels bioconjugated with shark antibodies (VNAR) for targeted drug delivery with potential applications in colon cancer therapies.

Cancer is the second leading cause of death worldwide. To combat this disease, novel and specialized therapeutic systems are urgently needed. This is the first study to explore a system that combines shark variable domain (Fv) of new antigen receptor (VNAR) antibodies (hereinafter VNARs), PEGylated nanogels (pH-sensitive poly(N,N-diethylaminoethyl methacrylate, PDEAEM), and the anticancer drug 5-fluorouracil (5-FU) to explore its potential applications in colon cancer therapies. Nanogels were functionalized in a scalable reaction with an N-hydroxysuccinimide (NHS)-terminated polyethylene glycol derivative and bioconjugated with shark antibodies. Dynamic light scattering measurements indicated the presence of monodispersed nanogels (74 to 236 nm). All systems maintained the pH-sensitive capacity to increase in size as pH decreased. This has direct implications for the release kinetics of 5-FU, which was released faster at pH 5 than at pH 7.4. After bioconjugation, the ELISA results indicated VNAR presence and carcinoembryonic antigen (CEA) recognition. In vitro evaluations of HCT-116 colon cancer cells indicated that functionalized empty nanogels are not cytotoxic and when loaded with 5-FU, the cytotoxic effect of the drug is preserved. A 15% reduction in cell viability was observed after two hours of contact with bioconjugated nanogels when compared to what was observed with non-bioconjugated nanogels. The prepared nanogel system shows potential as an effective and site-specific nanocarrier with promising applications in in vivo studies of colon cancer therapies.

Shark nanobodies with potent SARS-CoV-2 neutralizing activity and broad sarbecovirus reactivity

Despite rapid and ongoing vaccine and therapeutic development, SARS-CoV-2 continues to evolve and evade, presenting a need for next-generation diverse therapeutic modalities. Here we show that nurse sharks immunized with SARS-CoV-2 recombinant receptor binding domain (RBD), RBD-ferritin (RFN), or spike protein ferritin nanoparticle (SpFN) immunogens elicit a set of new antigen receptor antibody (IgNAR) molecules that target two non-overlapping conserved epitopes on the spike RBD. Representative shark antibody variable NAR-Fc chimeras (ShAbs) targeting either of the two epitopes mediate cell-effector functions, with high affinity to all SARS-CoV-2 viral variants of concern, including the divergent Omicron strains. The ShAbs potently cross-neutralize SARS-CoV-2 WA-1, Alpha, Beta, Delta, Omicron BA.1 and BA.5, and SARS-CoV-1 pseudoviruses, and confer protection against SARS-CoV-2 challenge in the K18-hACE2 transgenic mouse model. Structural definition of the RBD-ShAb01-ShAb02 complex enabled design and production of multi-specific nanobodies with enhanced neutralization capacity, and picomolar affinity to divergent sarbecovirus clade 1a, 1b and 2 RBD molecules. These shark nanobodies represent potent immunotherapeutics both for current use, and future sarbecovirus pandemic preparation.

ADR3, a next generation i-body to human RANKL, inhibits osteoclast formation and bone resorption

Osteoporosis is a chronic skeletal condition characterized by low bone mass and deteriorated microarchitecture of bone tissue, and puts tens of millions of people at high risk of fractures. New therapeutic agents like i-bodies, a class of next-generation single-domain antibodies, are needed to overcome some limitations of conventional treatments. An i-body is a human immunoglobulin scaffold with two long binding loops that mimic the shape and position of those found in shark antibodies, the variable new antigen receptors (VNARs) of sharks. Its small size (∼12 kDa) and long binding loops provide access to drug targets which are considered undruggable by traditional monoclonal antibodies. Here, we have successfully identified a human receptor activator of nuclear-factor κΒ (RANK) ligand (hRANKL) i-body, ADR3, which demonstrates high binding affinity to hRANKL with no adverse effect on the survival or proliferation of bone marrow-derived macrophages (BMMs). Differential scanning fluorimetry suggested that ADR3 was stable and able to tolerate a wide range of physical environments (including both temperature and pH). In addition, in vitro studies showed a dose-dependent inhibitory effect of ADR3 on osteoclast differentiation, podosome belt formation, and bone resorption activity. Further investigation on mechanism of action of ADR3 revealed that it could inhibit RANKL-mediated signaling pathways, supporting the in vitro functional observations. These clues collectively indicate that RANKL antagonist ADR3 attenuates osteoclast differentiation and bone resorption, with potential to serve as a novel therapeutic to protect against bone loss.

A Single Domain Shark Antibody Targeting the Transferrin Receptor 1 Delivers a TrkB Agonist Antibody to the Brain and Provides Full Neuroprotection in a Mouse Model of Parkinson's Disease.

Single domain shark antibodies that bind to the transferrin receptor 1 (TfR1) on brain endothelial cells have been used to shuttle antibodies and other cargos across the blood brain barrier (BBB) to the brain. For these studies the TXB4 brain shuttle was fused to a TrkB neurotrophin receptor agonist antibody. The TXB4-TrkB fusion retained potent agonist activity at its cognate receptor and after systemic administration showed a 12-fold increase in brain levels over the unmodified antibody. Only the TXB4-TrkB antibody fusion was detected within the brain and localized to TrkB positive cells in the cortex and tyrosine hydroxylase (TH) positive dopaminergic neurons in the substantia nigra pars compacta (SNc), where it was associated with activated ERK1/2 signaling. When tested in the 6-hydroxydopamine (6-OHDA) mouse model of Parkinson’s disease (PD), TXB4-TrkB, but not the unmodified antibody, completely prevented the 6-OHDA induced death of TH positive neurons in the SNc. In conclusion, the fusion of the TXB4 brain shuttle allows a TrkB agonist antibody to reach neuroprotective concentrations in the brain parenchyma following systemic administration.

Study review of camelid and shark antibodies for biomedical and biotechnological applications

The antibodies of camelids and sharks are about one–half of the conventional ones while regular antibodies have four protein chains: two light and two heavy, these small antibodies studied have just two heavy chains; they lack a light chain. In recent years, nanobodies have been the focus of attention because they can recognize epitopes that are usually not antigenic (hidden) for conventional antibodies. On the clinical side, researchers are testing nanobodies (Nbs) in the fight against diseases and disease diagnosis. Nanobodies also are attractive because they can prevent protein aggregation and clear the already existing aggregates. Furthermore, new treatments using these Nbs can neutralize the severe acute respiratory syndrome coronavirus (SARS-CoV-2) for preventing COVID-19. In this review, we sum up recent findings of the proposed nanobodies for their potential application.

Preliminary characterization of pathogen-detection activities of serum antibodies from the banded houndshark Triakis scyllium

Antibodies of cartilaginous fish are of scientific interest due to their phylogenetic position. In the present study, we developed antiserum against IgM of the banded houndshark, Triakis scyllium, and characterized binding activity of the IgM against fish pathogenic bacteria. Pentameric and monomeric IgM antibodies were separated by gel filtration chromatography using high performance liquid chromatography and SDS–PAGE. Antisera were developed by immunizing rabbits with unfractionated IgM antibodies separated by SDS–PAGE electrophoresis. Shark serum antibodies were found to have binding affinity for Aeromonas hydrophila, Vibrio anguillarum, Edwardsiella tarda, and Pseudomonas plecoglossicida antigens but not Lactococcus garvieae by enzyme-linked immunosorbent assay. We speculate the binding activities of shark antibodies may confer protection against certain bacterial pathogens.

Shark Antibody Variable Domains Rigidify Upon Affinity Maturation-Understanding the Potential of Shark Immunoglobulins as Therapeutics.

Sharks and other cartilaginous fish are the phylogenetically oldest living organisms that have antibodies as part of their adaptive immune system. As part of their humoral adaptive immune response, they produce an immunoglobulin, the so-called immunoglobulin new antigen receptor (IgNAR), a heavy-chain only antibody. The variable domain of an IgNAR, also known as VNAR, binds the antigen as an independent soluble domain. In this study, we structurally and dynamically characterized the affinity maturation mechanism of the germline and somatically matured (PBLA8) VNAR to better understand their function and their applicability as therapeutics. We observed a substantial rigidification upon affinity maturation, which is accompanied by a higher number of contacts, thereby contributing to the decrease in flexibility. Considering the static x-ray structures, the observed rigidification is not obvious, as especially the mutated residues undergo conformational changes during the simulation, resulting in an even stronger network of stabilizing interactions. Additionally, the simulations of the VNAR in complex with the hen egg-white lysozyme show that the VNAR antibodies evidently follow the concept of conformational selection, as the binding-competent state already preexisted even without the presence of the antigen. To have a more detailed description of antibody–antigen recognition, we also present here the binding/unbinding mechanism between the hen egg-white lysozyme and both the germline and matured VNARs. Upon maturation, we observed a substantial increase in the resulting dissociation-free energy barrier. Furthermore, we were able to kinetically and thermodynamically describe the binding process and did not only identify a two-step binding mechanism, but we also found a strong population shift upon affinity maturation toward the native binding pose.

Specific Targeting of Lymphoma Cells Using Semisynthetic Anti-Idiotype Shark Antibodies.

The B-cell receptor (BCR) is a key player of the adaptive immune system. It is a unique part of immunoglobulin (Ig) molecules expressed on the surface of B cells. In case of many B-cell lymphomas, the tumor cells express a tumor-specific and functionally active BCR, also known as idiotype. Utilizing the idiotype as target for lymphoma therapy has emerged to be demanding since the idiotype differs from patient to patient. Previous studies have shown that shark-derived antibody domains (vNARs) isolated from a semi-synthetic CDR3-randomized library allow for the rapid generation of anti-idiotype binders. In this study, we evaluated the potential of generating patient-specific binders against the idiotype of lymphomas. To this end, the BCRs of three different lymphoma cell lines SUP-B8, Daudi, and IM-9 were identified, the variable domains were reformatted and the resulting monoclonal antibodies produced. The SUP-B8 BCR served as antigen in fluorescence-activated cell sorting (FACS)-based screening of the yeast-displayed vNAR libraries which resulted after three rounds of screening in the enrichment of antigen-binding vNARs. Five vNARs were expressed as Fc fusion proteins and consequently analyzed for their binding to soluble antigen using biolayer interferometry (BLI) revealing binding constants in the lower single-digit nanomolar range. These variants showed specific binding to the parental SUP-B8 cell line confirming a similar folding of the recombinantly expressed proteins compared with the native cell surface-presented BCR. First initial experiments to utilize the generated vNAR-Fc variants for BCR-clustering to induce apoptosis or ADCC/ADCP did not result in a significant decrease of cell viability. Here, we report an alternative approach for a personalized B-cell lymphoma therapy based on the construction of vNAR-Fc antibody-drug conjugates to enable specific killing of malignant B cells, which may widen the therapeutic window for B-cell lymphoma therapy.

Abstract 2651: Characterization of TIE2 function in cancer cell dormancy and bone metastases

Abstract After arriving in bone, cancer cells can enter a dormancy state that can last for years, during which they are resistant to treatment. The bone microenvironment induces dormancy via mechanisms similar to the ones of hematopoietic stem cells (HSC). The angiopoietin receptor, TIE2, induces the dormancy of HSC and some cancer cells and is a target for anti-angiogenic treatments. However, its inhibition may restart the proliferation of cancer cells, posing the risk of bone metastasis growth, while increasing the sensitivity of cancer cells to chemotherapy. Thus, we aim to characterize the role of TIE2 in dormancy of bone metastases. We found that higher levels of Tie2 in the primary tumor of breast cancer (BCa) patients are associated with a good prognosis in multiple cohorts. However, expression of Tie2 protein or mRNA was either very low or undetected in cultured BCa and prostate cancer (PCa) cell lines. BCa and PCa cells transduced to express TIE2 constitutively showed a gradual loss of TIE2+ cells, despite continuous antibiotic selection. Congruently, in 5 different patient datasets, Tie2 expression is significantly lower in invasive BCa compared to healthy breast tissue. These results suggest that there is no expression of TIE2 in growing cancer cells. In MCF-7 TIE2tet BCa cells conditional TIE2 expression with a Tet-On system caused a significant decrease of cell proliferation and the percentage of cells in S and G2/M phase but not in MCF-7 GFPtet control cells. Expression of TIE2, but not GFP, significantly decreased mRNA levels of the proliferation marker Ki67 while increasing the dormancy markers p21 and p27. Additionally, TIE2 expression increased the resistance to the chemotherapeutic 5-Fluorouracil. 4T1 Tie2tet cells inoculated in the mammary fat pads of Balb/C mice receiving Dox showed significantly reduced tumor volume and weight of excised tumors compared to control mice, suggesting TIE2 caused a growth reduction. When intracardiacally inoculated, 4T1 Tie2tet cells caused bone metastases regardless of TIE2 expression. However, the osteolysis area was significantly decreased when TIE2 was expressed, indicating a delayed bone metastasis development. To reverse dormancy and increase chemotherapy sensitivity, we selected a shark antibody (vNAR) that binds to TIE2 using a synthetic library and phage display. In silico modeling indicated this clone could bind to the FN2 domain and inhibit the multimerization of TIE2, which could prevent signaling. Overall, we found that expression of TIE2 decreases BCa cell growth in vitro and in vivo, and their sensitivity to 5-Fluorouracil, while increasing the expression of dormancy marker in vitro, and the time to the development of metastasis and survival of BCa patients. Our results are then consistent with TIE2 inducing dormancy of cancer cells. Thus, neutralizing TIE2 with a vNAR in combination with cancer therapy could be used for the treatment of patients at risk of bone metastases. Citation Format: Florian Drescher, Salvador Dueñas, Felipe Olvera-Rodriguez, Nicolás Serafín Higuera, Samanta Jiménez Flores, Patricia Juarez, Alexei Licea-Navarro, Pierrick G. Fournier. Characterization of TIE2 function in cancer cell dormancy and bone metastases [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2651.

Brain delivery of biologics using a cross-species reactive transferrin receptor 1 VNAR shuttle.

Transferrin receptor 1 (TfR1) mediated transcytosis is an attractive strategy to enhance brain uptake of protein drugs, but translation remains a challenge. Here, a single domain shark antibody VNAR fragment (TXB2) with similar affinity to murine and human TfR1 was used to shuttle protein cargo into the brain. TXB2 was fused to a human IgG1 Fc domain (hFc) or to the amyloid-β (Aβ) antibody bapineuzumab (Bapi). TXB2-hFc displayed 20-fold higher brain concentrations compared with a control VNAR-hFc at 18hours post-injection in wt mice. At the same time point, brain concentrations of Bapi-TXB2 was threefold higher than Bapi. In transgenic mice overexpressing human Aβ, the brain-to-blood concentration ratio increased with time due to interaction with intracerebral Aβ deposits. The relatively stable threefold difference between Bapi-TXB2 and Bapi was observed for up to 6days after injection. PET imaging and ex vivo autoradiography revealed more parenchymal distribution of Bapi-TXB2 compared with Bapi. In conclusion, the TXB2 VNAR shuttle markedly increased brain uptake of protein cargo and increased brain concentrations of the Aβ binding antibody Bapi.

Selection and Characterization of Anti-idiotypic Shark Antibody Domains.

The antibody repertoire of cartilaginous fish comprises an additional heavy-chain-only antibody isotype that is referred to as IgNAR (immunoglobulin novel antigen receptor). Its antigen-binding site consists of one single domain (vNAR) that is reportedly able to engage a respective antigen with affinities similar to those achieved by conventional antibodies. While vNAR domains offer a reduced size, which is often favorable for applications in a therapeutic as well as a biotechnological setup, they also exhibit a high physicochemical stability. Together with their ability to target difficult-to-address antigens such as virus particles or toxins, these shark-derived antibody domains seem to be predestined as tools for biotechnological and diagnostic applications. In the following chapter, we will describe the isolation of anti-idiotypic vNAR domains targeting monoclonal antibody paratopes from semi-synthetic, yeast-displayed libraries. Anti-idiotypic vNAR variants could be employed for the characterization of antibody-based therapeutics (such as antibody-drug conjugates) or as positive controls in immunogenicity assays. Peculiarly, when using semi-synthetic vNAR libraries, we found that it is not necessary to deplete the libraries using unrelated antibody targets, which enables a fast and facile screening procedure that exclusively delivers anti-idiotypic binders.

CROSSBETA STABLE Aβ OLIGOMERS: DISEASE-RELEVANT DRUG TARGETS AND VERSATILE REFERENCE STANDARDS IN BIOMARKER ASSAYS IN AD

There is accumulating evidence that soluble amyloid beta (Aβ) oligomers, rather than the amyloid fibrils, are the principal pathogenic species in Alzheimer disease (AD). Because they exhibit acute synaptic toxicity and induce neurodegenerative processes, and because the Aβ oligomer levels in cerebrospinal fluid appear to correlate with disease severity and progression, these species constitute an optimal drug target and possible useful diagnostic marker in early AD. Unfortunately, the instability and limited characterization of oligomer preparations have seriously impeded research and development in this field. Crossbeta has addressed this problem by developing a technology to reproducibly generate stable soluble Aß1–42 oligomers that are devoid of monomeric or fibrillar Aß and show biophysical properties indistinguishable from the native species. Furthermore, Crossbeta oligomers demonstrate typical functionality of pathological Aβ species such as neurotoxicity, in vivo LTP depression and induction of inflammatory response. The availability of the stable oligomers opens previously inaccessible avenues for R&D. For example, it enabled high throughput screening of drug-like compound libraries. Derived from a 100k compound screening effort, small molecule CBB68 was identified and shown to neutralize oligomer-induced neurotoxicity in rat primary neurons and to rescue the synaptic deficit induced by Aβ oligomers in vivo as well as in vitro. In particular, the in vivo LTP assay demonstrated the efficacy and brain penetration of CBB68 after intravenous administration. In another application, Crossbeta's oligomers have also been used to select shark antibodies that specifically recognize a conformational oligomeric epitope and bind to Aß1–42 oligomers with high affinity (sub-nM), but not to the monomers or fibrils. This antibody provides potential to selectively quantify Aß1–42 oligomers in patient material as part of a high sensitivity biomarker assay together with the stable Crossbeta oligomers as calibrator. Furthermore, the oligomers are excellent research tools, e.g. in disease-relevant bioassays. We show and conclude that the stable oligomers enable multiple applications and constitute optimum drug targets.

The structural analysis of shark IgNAR antibodies reveals evolutionary principles of immunoglobulins.

Sharks and other cartilaginous fish are the phylogenetically oldest living organisms that rely on antibodies as part of their adaptive immune system. They produce the immunoglobulin new antigen receptor (IgNAR), a homodimeric heavy chain-only antibody, as a major part of their humoral adaptive immune response. Here, we report the atomic resolution structure of the IgNAR constant domains and a structural model of this heavy chain-only antibody. We find that despite low sequence conservation, the basic Ig fold of modern antibodies is already present in the evolutionary ancient shark IgNAR domains, highlighting key structural determinants of the ubiquitous Ig fold. In contrast, structural differences between human and shark antibody domains explain the high stability of several IgNAR domains and allowed us to engineer human antibodies for increased stability and secretion efficiency. We identified two constant domains, C1 and C3, that act as dimerization modules within IgNAR. Together with the individual domain structures and small-angle X-ray scattering, this allowed us to develop a structural model of the complete IgNAR molecule. Its constant region exhibits an elongated shape with flexibility and a characteristic kink in the middle. Despite the lack of a canonical hinge region, the variable domains are spaced appropriately wide for binding to multiple antigens. Thus, the shark IgNAR domains already display the well-known Ig fold, but apart from that, this heavy chain-only antibody employs unique ways for dimerization and positioning of functional modules.

Construction of chimeric shark antibody fragment binding p24 nucleocapsid protein for detection of antibodies for immunodeficiency virus type 1 in serum using hemagglutination

Background: It has been studied that shark antibodies or antibody fragments are important tools for experimental research and medical application. The advantages of using these sorts of molecules are their small size, high solubility and good tissue penetration in vivo. A chimeric shark antibody fragment binding p24 nucleocapsid protein was successfully constructed for HIV detection. The aim of this research was to determine the presence of antibodies for immunodeficiency virus type 1 in serum through ex vivo hemagglutination. Methods: Using a shark gene that codifies a antibody fragment capable of recognising glycophorin A from erythrocytes membrane, was obtained from a non immune shark library. The gene was bind with p24 gene using a double cloning method in order to construct a chimeric gene. A recombinant chimeric protein was produced cloning the chimeric gene in an expression vector through Escherichia coli. The effectiveness of the chimeric protein was evaluated carrying out hemagglutination assays. Erythrocytes free of plasma and serum were subjected to a series of wash phosphate buffers and 38 HIV positive serum samples donated by the CAPASITS clinic, Tijuana B. C. Mexico, were analysed. Results: The p24 gen was isolated from clinical samples, amplified and ligated to both the vector carrier and the shark gene. An enzyme-linked immunobsorbent assay (ELISA) was performed to the chimeric protein with the aim of verifying the presence of p24 and recognition of glycophorins. Once the functionality of the chimeric proteinwas confirmed, the38HIVpositive serumsamples were submitted to hemagglutination assays. Positive results were obtained in 36 samples out of 38,which represent 95% of efficiency. Conclusion: These findings indicate the efficiency of using this chimeric protein for detection of antibodies for immunodeficiency virus type 1 in serum and the possibility to carry out further research inwhole blood samples aswell as anotherHIV type. Also, a viable potential HIV-1 rapid diagnosis test, aiming it to rural clinical practice for it is easy to perform without any special equipment.

Evolution of an Interloop Disulfide Bond in High-Affinity Antibody Mimics Based on Fibronectin Type III Domain and Selected by Yeast Surface Display: Molecular Convergence with Single-Domain Camelid and Shark Antibodies

The 10th human fibronectin type III domain ( 10Fn3) is one of several protein scaffolds used to design and select families of proteins that bind with high affinity and specificity to macromolecular targets. To date, the highest affinity 10Fn3 variants have been selected by mRNA display of libraries generated by randomizing all three complementarity-determining region -like loops of the 10Fn3 scaffold. The sub-nanomolar affinities of such antibody mimics have been attributed to the extremely large size of the library accessible by mRNA display (10 12 unique sequences). Here we describe the selection and affinity maturation of 10Fn3-based antibody mimics with dissociation constants as low as 350 pM selected from significantly smaller libraries (10 7–10 9 different sequences), which were constructed by randomizing only 14 10Fn3 residues. The finding that two adjacent loops in human 10Fn3 provide a large enough variable surface area to select high-affinity antibody mimics is significant because a smaller deviation from wild-type 10Fn3 sequence is associated with a higher stability of selected antibody mimics. Our results also demonstrate the utility of an affinity-maturation strategy that led to a 340-fold improvement in affinity by maximizing sampling of sequence space close to the original selected antibody mimic. A striking feature of the highest affinity antibody mimics selected against lysozyme is a pair of cysteines on adjacent loops, in positions 28 and 77, which are critical for the affinity of the 10Fn3 variant for its target and are close enough to form a disulfide bond. The selection of this cysteine pair is structurally analogous to the natural evolution of disulfide bonds found in new antigen receptors of cartilaginous fish and in camelid heavy-chain variable domains. We propose that future library designs incorporating such an interloop disulfide will further facilitate the selection of high-affinity, highly stable antibody mimics from libraries accessible to phage and yeast surface display methods.

RNA mutagenesis yields highly diverse mRNA libraries for in vitro protein evolution

BackgroundIn protein drug development, in vitro molecular optimization or protein maturation can be used to modify protein properties. One basic approach to protein maturation is the introduction of random DNA mutations into the target gene sequence to produce a library of variants that can be screened for the preferred protein properties. Unfortunately, the capability of this approach has been restricted by deficiencies in the methods currently available for random DNA mutagenesis and library generation. Current DNA based methodologies generally suffer from nucleotide substitution bias that preferentially mutate particular base pairs or show significant bias with respect to transitions or transversions. In this report, we describe a novel RNA-based random mutagenesis strategy that utilizes Qβ replicase to manufacture complex mRNA libraries with a mutational spectrum that is close to the ideal.ResultsWe show that Qβ replicase generates all possible base substitutions with an equivalent preference for mutating A/T or G/C bases and with no significant bias for transitions over transversions. To demonstrate the high diversity that can be sampled from a Qβ replicase-generated mRNA library, the approach was used to evolve the binding affinity of a single domain VNAR shark antibody fragment (12Y-2) against malarial apical membrane antigen-1 (AMA-1) via ribosome display. The binding constant (KD) of 12Y-2 was increased by 22-fold following two consecutive but discrete rounds of mutagenesis and selection. The mutagenesis method was also used to alter the substrate specificity of β-lactamase which does not significantly hydrolyse the antibiotic cefotaxime. Two cycles of RNA mutagenesis and selection on increasing concentrations of cefotaxime resulted in mutants with a minimum 10,000-fold increase in resistance, an outcome achieved faster and with fewer overall mutations than in comparable studies using other mutagenesis strategies.ConclusionThe RNA based approach outlined here is rapid and simple to perform and generates large, highly diverse populations of proteins, each differing by only one or two amino acids from the parent protein. The practical implications of our results are that suitable improved protein candidates can be recovered from in vitro protein evolution approaches using significantly fewer rounds of mutagenesis and selection, and with little or no collateral damage to the protein or its mRNA.

Structure of a shark IgNAR antibody variable domain and modeling of an early‐developmental isotype

The new antigen receptor (IgNAR) antibodies from sharks are disulphide bonded dimers of two protein chains, each containing one variable and five constant domains. Three types of IgNAR variable domains have been discovered, with Type 3 appearing early in shark development and being overtaken by the antigen-driven affinity-matured Type 1 and 2 response. Here, we have determined the first structure of a naturally occurring Type 2 IgNAR variable domain, and identified the disulphide bond that links and stabilizes the CDR1 and CDR3 loops. This disulphide bridge locks the CDR3 loop in an "upright" conformation in contrast to other shark antibody structures, where a more lateral configuration is observed. Further, we sought to model the Type 3 isotype based on the crystallographic structure reported here. This modeling indicates (1) that internal Type 3-specific residues combine to pack into a compact immunoglobulin core that supports the CDR loop regions, and (2) that despite apparent low-sequence variability, there is sufficient plasticity in the CDR3 loop to form a conformationally diverse antigen-binding surface.

A shark antibody heavy chain encoded by a nonsomatically rearranged VDJ is preferentially expressed in early development and is convergent with mammalian IgG.

In most vertebrate embryos and neonates studied to date unique antigen receptors (antibodies and T cell receptors) are expressed that possess a limited immune repertoire. We have isolated a subclass of IgM, IgM(1gj), from the nurse shark Ginglymostoma cirratum that is preferentially expressed in neonates. The variable (V) region gene encoding the heavy (H) chain underwent V-D-J rearrangement in germ cells ("germline-joined"). Such H chain V genes were discovered over 10 years ago in sharks but until now were not shown to be expressed at appreciable levels; we find expression of H(1gj) in primary and secondary lymphoid tissues early in life, but in adults only in primary lymphoid tissue, which is identified in this work as the epigonal organ. H(1gj) chain associates covalently with light (L) chains and is most similar in sequence to IgM H chains, but like mammalian IgG has three rather than the four IgM constant domains; deletion of the ancestral IgM C2 domain thus defines both IgG and IgM(1gj). Because sharks are the members of the oldest vertebrate class known to possess antibodies, unique or specialized antibodies expressed early in ontogeny in sharks and other vertebrates were likely present at the inception of the adaptive immune system.

Antibody production in sharks and humans: A role for natural antibodies

Although gene segments specifying Igs of all vertebrates show clear homology, their arrangements differ markedly, thereby suggesting that the mechanisms for the generation of diversity and for the regulation of gene expression may be quite distinct. In the sandbar shark, light chain gene segments are distributed as apparently independent clusters consisting of V, J, and C elements that require rearrangement for expression. The usual distance between V and C in the clusters is 3 kb but larger clusters occur. The V, J, and C elements are clearly homologous to those of human λ chains. Shark Igs resemble mammalian IgM in structure and gene similarity. IgM may comprise as much as 50% of serum proteins in the shark. By contrast, IgM in humans comprises less than 5%. Human autoantibodies usually are IgM. These show little dependence on thymic function for expression and tend to increase with age. We have carried out a study of the capacity of Igs of unimmunized sharks and people (normals and patients suffering from autoimmune diseases) to react against a panel of antigens, including those usually considered autoantibodies, such as thyroglobulin and single-stranded DNA. Sharks and humans possess IgM antibodies that react with thyroglobulin and ssDNA. Affinity-purified natural shark antibodies to thyroglobulin or ssDNA constitute small fractions of total IgM. They illustrate extensive cross-reactivity comparable to that shown by polyspecific IgM autoantibodies produced by human B cells (CD5+) that appear early in ontogeny.

Phylogeny of Immunoglobulin Structure and Function. XV. Idiotypic Analysis of Shark Antibodies

Nurse shark antibodies to the streptococcal A-variant carbohydrate were specifically purified from the sera of four individual animals and used as immunogens in guinea pigs. The resultant guinea pig antisera contained antibodies with apparent idiotypic specificities for the homologous shark proteins. The shark idiotypic sites were located on the Fab fragments and appeared to require the participation of H and L chains for full expression. Tests for cross reactivity employing the four guinea pig anti-idiotypic sera and antibodies from 13 immunized sharks were positive in only two cases (heterologous inhibition). These findings indicate that the idiotypic library (and by inference the antibody combining site reportoire) of nurse sharks to the streptococcal A-variant antigen is probably quite extensive.